Disease processes in the back of the eye result in pathologic changes that affect the physiology and function of the RPE/retina interface and can lead to new blood vessel formation (neovascularization or angiogenesis), retinal edema, and subsequent retinal detachment retinal detachments; age-related macular degeneration (AMD) in the elderly (60 yrs or older); retinopathy of prematurity (infants); diabetic retinopathy (in adults). Use of animal models helps us understand the role of retinal pigment epithelium (RPE) cells in visual physiology and pathology, and allows us to develop intervention strategies against degenerative eye diseases. Based on in vitro findings, in animal experiments using KO mice models we have studied the fine tuning of RPE physiology by microRNAs (miRNA) whose interactive networks regulate multiple cellular interactions and RPE function. From in vitro studies, we know that miRNAs regulate a significant portion of the transcriptome. The miRNAs are highly conserved across many species, helping us better understand their functional significance. Several miRNAs were identified whose expression is relative high in hfRPE compared to its adjacent tissues, including retina and choroid. These hfRPE-enriched miRNAs are miR-184, miR-187, miR-200, miR-221/222, miR-204, and miR-211, and several of them were found to be critical to the total tissue resistance of the hfRPE, suggesting their importance in RPE barrier function. To carry out this study, we produced a miR-204 knockout (KO) mouse line by a gene-targeting approach. The miR-204 gene was completely ablated and replaced with a neomycin resistant gene expression cassette via homologous recombination. Loss of miR-204 expression was determined in tissues normally enriched in miR-204, such as eye and brain, by Northern hybridization. The knockout mice are viable and do not exhibit gross developmental defects. However, OCT analysis of adult eye showed an irregular vasculature in retina and an extra mass protruding from the lens epithelium in 2/3rds of the knockout mice. Their physiological function in the eye is likely compromised since ERGs obtained from these mice showed a-wave and b-wave amplitude responses that were diminished in rod photoreceptors compared to control (n = 3-5). Additional functional studies (OCT, ERG) will be required to characterize the retinal defects and future studies will determine the possible structural alteration in eyes of miR-204 KO mice and identity the miR-204 target genes responsible for the physiological changes. In other sets of experiments we studied miR-155, an inflammatory miRNA, which was found to be enriched in choroid compared to RPE and retina. Its expression in RPE can be increased by a well-defined pro-inflammatory cytokine cocktail. Its physiological significance has been extensively studied in monocytes, macrophages, and dendritic cells where pro-inflammatory stimuli, such as bacterial lipopolysaccharide, polyinosinic:polycytidylic acid, or IFN increased miR-155 expression and miR-155 was shown to play an important role in inflammation. We are performing dark and light adapted ERGs on 3, 6, 9, 12, and 24 month old miR-204 and mir-155 KO mice to understand the role of these miRNAs in rod and cone photoreceptors as they progressively age. Additionally, dc-ERGs have been performed on these KO mice, and these dc-ERGs are a very powerful tool to visualize RPE function through monitoring of electrophysiological responses. The dc-ERG consists of three parts, a positive c-wave comprised of RPE apical membrane hyperpolarization due to increased K+ conductance and slow PIII response of the Muller cells, a negative fast oscillation (FO) comprised of basal membrane hyperpolarization due to reduced conductance of CFTR-mediated Cl channels, and positive light peak (LP) comprised of basal membrane depolarization by increased activity of Ca2+ -activated Cl channels. Thus, the dc-ERG enables us to isolate the electrical responses of the RPE from the rod and cone photoreceptors since the latter can be analyzed by recording normal retinal ERG a- and b-wave responses. The nm3342 mouse may be the first clinically appropriate animal model for studying serous detachment and central serious retinopathy (CSR). An early goal of our study was to determine gene differences in the RPE and choroid between mutant and wild type mice that could disrupt regulation and allow for fluid leakage. In these mutants, the choroidal or RPE cells may contain abnormal tight junctions that disrupt the seal that separates the extracellular spaces on either side of the RPE, or there may be RPE cell death. The mutation itself may cause abnormalities in the neural retina or the abnormalities only arise after retinal detachment. Rhegmatogenous detachment (where fluid flows through a hole or break in the retina) creates rapid cellular changes in the retina. Whether serous detachments cause similar changes is unknown. Current observations in human patients with CSR suggest degenerative changes in the photoreceptor layer, but their nature is unknown. We determined that photoreceptor degeneration and cell death occurred in the mice along with neuronal or glial remodeling which suggest mechanisms for vision loss in human CSR patients. Comparisons to gene expression in rhegmatogenous detachments of the same duration will be important as well. We have obtained the time-line of ocular structural changes in nm3342 mice by high-resolution OCT, a powerful non-invasive technique that provides additional structural data and help guide the cellular characterization of nm3342. Pilot studies will be carried out to determine if previously tested small molecules resolve detachment in the nm3342 mice, and we will begin exploring methods for the use of primary cell cultures of nm3342 RPE cells. These experiments should help us understand the special characteristics of serous retinal detachments, an important first step to understanding CSR in humans. In a separate set of experiments, based on our previous knowledge of retinal reattachment techniques (Maminishkis et al., IOVS, 2003), we developed a rat model to test various RPE transplants in preparation for clinical trials expected to begin in 2018. This technique was used in toxicology studies by COVANCE Laboratories to test systemic and local toxicology of caused by transplanted iPS-derived RPE cells. This lead to the generation of preclinical data required for our IND submission to the FDA for our upcoming AMD clinical trial (2018). The successful transplantation of iPSC-derived RPE on scaffolds in humans is a procedure that required the development of surgical techniques and tools using the swine pre-clinical animal model. Based on this pre-clinical work we can directly transpose the usage of these transplantation tools to the upcoming 2018 human clinical trials.